The present invention is directed to a method and apparatus for automatically unloading an isoelectric focusing gel from a tube onto a surface, and particularly a gel slab. More particularly, the invention is directed to a method and apparatus for unloading a gel from a tube as a continuous bead.
Genomes provide the sequence information required to construct proteins that are the working parts of living cells. Genomes and genes are linear constructs composed of four different nucleotides arranged in triplet condons that specify the order and identity of the approximately 20 different amino acids that make up proteins. The nucleic acids are chemically very similar, and are arranged in very long contiguous sequences with intervening non-coding regions. For analysis, nucleic acids must be cut up into fragments of tractable length using shearing forces or restriction enzymes which cut the nucleic acid at specific known sites.
Proteins are made of amino acid subunits that have a range of different isoelectric points, molecular weights, and solubility or hydrophobicity characteristics. The synthesized peptides have exactly defined lengths, and roll up or are assembled into proteins of well defined molecular weights. The estimated 100,000 different primary proteins in man have a range of charge densities and isoelectric points, solubilities, and surface characteristics not found in nucleic acids. Further, proteins have a range of surface conformations which mediate specific interactions between proteins, between proteins and nucleic acids, and, in the form of enzymatically active sites, between low molecular weight metabolites, and all the various types of macromolecules found in cells and foodstuffs. Proteins are the molecular machines that carry out the panoply of syntheses, disassemblies and degradations, immunochemical defense reactions, and paratactic interactions that underlie the assembly of membranes and subcellular organelles.
There is a need for analytical methods that allow a large fraction of the total number of proteins present in a cell or tissue to be detected and quantitated. The quantitative analysis of large sets of proteins that have such a wide variety of functions, sizes, conformation, activities, solubilities, and charge characteristics is both a centrally important challenge, and an exceedingly difficult problem. The problem is rendered even more difficult by the requirement that analysis detecting thousands of proteins per analyses be done in parallel on relatively large numbers of samples in a reasonable time to do experimental toxicological and pharmacological studies.
The electrophoretic mobility of a non-denatured protein is a function of the surface charges of either the monomeric protein or the sum of the surface charges of the subunits, and these are generally used under rate-zonal conditions, i.e., under conditions where the proteins move through a gel or other support at one pH. The distance traveled is a function of the charge to mass ratio, and a function of electrophoresis time. Second dimension separations are done in gradient gels of decreasing pore size such that proteins move until movement essentially ceases as the protein reach pore sizes that prevent further movement. Experimental attempts to develop two dimensional methods based on these parameters using non-denaturing conditions have not yielded the resolution required.
Two-dimensional methods involving denaturing conditions have been explored and widely adopted. The initial separation is done in concentrated urea in the presence of ampholytes which are a heterogeneous mixture of synthetic polymers having wide variation in the ratio of acidic to basic groups. When these are subjected to an electrical field in a gel, the ampholytes sort themselves out into a continuous series based on the isoelectric point. Proteins move along the gel until they reach their own isoelectric point and stop. Further, since the proteins are denatured and unrolled, their isoelectric points reflect the sum of all of the charged groups in the protein, whether previously external or internal in the native state. The isoelectric point determination in such a separation can be calculated from the amino acid composition of the protein, and is a valuable parameter for protein classification.
The second dimensional separation is based on the length (and hence the mass) of the unrolled denatured protein and takes place in the following way. Proteins from the isoelectric separation are exposed to a highly charged detergent which has attached the longest paraffin chain which will remain extended in solution, and not fold back on itself. Sodium dodecyl sulfate (SDS) is the detergent of choice, and in solution will uniformly coat unrolled polypeptide chains, and attach to them by hydrophobic linkages, leaving the highly charged sulfate groups on the surface. The result is particles of approximately rod shape having approximately equal charge-to-mass ratios. Particles having equal charge-to-mass ratios move at the same rate in electrical fields, so that all proteins covered with SDS should have equal mobility in solution. However, if electrophoresis of such particles is done in a microporous gel, then larger particles will be retarded relative to smaller ones.
In practice, the resolutions of these two separate methods are quite high. At least 150 proteins can be resolved from a suitable mixture by isoelectric focusing, and an equal number resolved from a suitable protein mixture by SDS electrophoresis. If the two processes can be mated together in a two-dimensional array, the final resolution should be the product of the resolution of the two methods separately, i.e., 1502 or 22,500. Experimentally, as many as 5,000 proteins have been resolved in large two-dimensional electrophoresis gels, and the theoretical resolution of current electrophoresis as calculated from spot sizes, and the number of spots which could theoretically be packed into the gel area used is around 30,000.
It is quite evident that a key step in the high-resolution two-dimensional electrophoresis technique using isoelectric focusing followed by SDS electrophoresis in the second dimension is mating the two methods together without the loss of resolution inherent in collecting and separately analyzing fractions.
Experimentally, isoelectric focusing is done under temperature controlled conditions in glass tubes (ISO tubes) having an internal diameter of approximately 0.5-2 mm, and approximately 30 cm long. ISO tubes are then attached to a small syringe full of water or buffer solution, and the gels extruded by hand along the top of a second-dimension gel cast between two glass plates. An empty space is typically formed between the top of the gel and the top of the plates. The gels are carefully extruded into this space by a double movement in which the syringe plunger is moved to extrude the gel as the ISO tube containing the gel is moved laterally along the top of the second dimension gel. This movement requires considerable skill, and many gels are broken as they are extruded and moved into place. It is further evident that different portions of the extruded gels may be stretched differently, causing distortion in the final 2D pattern. A further difficulty is that this step is the most variable and time consuming one in present programs aimed at automating the entire 2D process, in which batches of analyses varying from 10-60 are run in parallel. The 2D protein analysis has become a core analytical method in pharmacology and toxicology, and mass spectrometric analysis and identification of proteins in spots from 2D gels has become routine and essential. Accordingly, there is a continuing need in the industry for a system and method for automatically unloading large sets of gels from isoelectric focusing gel tubes directly onto second dimension gels with minimal distortion or breakage.
The present invention is directed to a method and apparatus for unloading a gel from a tube. More particularly, the invention is directed to a method and apparatus for the unloading of an isoelectric gel from a gel tube onto a gel slab or other work surface.
Accordingly, a primary object of the invention is to provide a method and apparatus for unloading a substance, and particularly a gel, from a tube substantially without distortion of the substance.
A further object of the invention is to provide a method and apparatus for unloading an isoelectric gel from a gel tube in a substantially uniform and controlled manner.
Another object of the invention is to provide an apparatus for removing a gel body from a cylindrical tube substantially without compressing or elongating the gel body.
A further object of the invention is to provide an apparatus for removing a substance from a cylindrical tube onto a surface by passing a plunger rod through the tube at a substantially uniform speed.
A further object of the invention is to provide a method and apparatus for unloading a gel from a cylindrical tube using a plunger rod mounted in a fixed position where one end of the tube slides onto the plunger rod to discharge the gel from the opposite end of the tube.
Another object of the invention is to provide a method and apparatus for unloading a substance from a tube at a controlled rate where the tube is moved along the surface at a controlled rate to discharge of the gel from the tube and uniformly onto a surface.
A further object of the invention is to provide a method and apparatus for discharging a gel from a tube using a flexible plunger member having a diameter greater than an internal diameter of the tube where the plunger member is fitted into one end of the tube and pushed along the length of the tube.
Still another object of the invention is to provide an apparatus for unloading a gel from a tube onto a surface where the apparatus moves the tube across the surface at a substantially constant speed and constant angle with respect to the direction of movement of the tube.
A further object of the invention is to provide an apparatus for unloading a gel from a plurality of tubes where the apparatus includes a movable support for the tubes, a plunger rod associated with each tube, and a stationary support coupled to one end of the rods, where the movable support moves toward the stationary support to slide one end of the tubes over the respective plunger rod and to unload the gel from the opposite end of the tubes.
The objects and advantages of the invention are basically attained by providing an apparatus for unloading a substance from a tube. The apparatus comprises a first support assembly for supporting a plurality of tubes, each of the tubes having an axial passage, a first open end and a second open end, the first end of the tube being coupled to the first support; a second support spaced from the first support; a plurality of plungers having a first end coupled to the second support and a second end axially aligned with an axial passage of a respective tube; and a drive assembly for moving the first support along a linear path toward the second support, whereby the tubes slide onto the respective plunger to unload the substance from the second end of the tubes.
The objects and advantages of the invention are further attained by providing an apparatus for unloading a substance from a tube onto a surface. The apparatus comprises a first support having a first side and a second side with at least one aperture extending through the carriage between the first side and the second side and having a removable carriage; a tube support member for supporting at least one tube containing the substance, the tube support member being coupled to the first side of the first support so that the tube is aligned with the at least one aperture; a second support spaced from the first support; at least one plunger rod having a first end coupled to the second support and a second end received in the at least one aperture of the first support; and a drive assembly for moving the carriage, in a linear path toward the second support whereby the plunger rod passes through the tube to unload the substance onto a surface.
The objects and advantages of the invention are still further attained by providing an apparatus for unloading an electrophoresis gel from an electrophoresis gel tube onto a gel slab. The apparatus comprises a first support having a first side and a second side with a plurality of apertures extending through the first support between the first side and the second side and having a movable carriage; a gel tube member having a plurality of electrophoresis gel tubes containing the electrophoresis gel, the gel tubes having a first end coupled to the gel tube support member and a second end spaced from the first support member, the gel tube support member being coupled to the first side of the first support so that the tubes are aligned with a respective aperture; a second support spaced from the first support; a plurality of plunger rods having a first end coupled to the second support and a second end received in the at least one aperture of the first support; a plurality of vertically oriented gel slabs having a top edge aligned with a respective gel tube; and a drive assembly for moving the carriage toward the second support whereby the plunger rod passes through the tube to unload the electrophoresis gel onto the top edge of the gel slabs.
The objects and advantages of the invention are yet further attained by providing an apparatus for unloading a gel from an isoelectric focusing gel tube. The apparatus comprises a housing having a first end, a second end opposite the first end and a side wall,. The housing has an axial passage extending between the first and second ends. The axial passage has a first open end at the first end of the housing and a second open end at the second end of the housing. A plunger rod has a first end positioned in the first open end of the axial passage of the housing. A gel tube has an axial bore containing an isoelectric focusing gel. The gel tube has a substantially cylindrical shape with a first open axial end and a second open axial end. The first open axial end of the gel tube is positioned in the second open end of the axial passage. A resilient plunger member is positioned between the first end of the plunger rod and the gel within the gel tube. The resilient plunger member has an outer dimension to fit within the bore of the gel tube.
The objects and advantages of the invention are still further attained by providing a method of unloading an isoelectric focusing gel from a gel tube. The method comprises providing a gel tube having an axial bore containing an isoelectric focusing gel. The gel tube has a first open axial end and a second open axial end. The first end of the gel tube is coupled to a first end of an unloading assembly. The unloading assembly has a flexible plunger member aligned with the first open axial end of the gel tube and a reciprocating plunger rod aligned with the plunger member and the first open axial end of the gel tube. The plunger rod moves against the plunger member and forces the plunger member and the first end of the plunger rod through the axial passage of the gel tube to unload the gel.
The objects, advantages and salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention in conjunction with the annexed drawings which form a part of this original disclosure.